The new continuous cell line HOK-I derived from a grade-III transitional-cell bladder carcinoma with foci of squamous and glandular differentiation was shown to retain this pheno-typical heterogeneity for more than 45 passages in vitro. Electron microscopy revealed transitional as well as a considerable proportion of squamous carcinoma and adenocarcinoma cells. PAS-positive mucus was detected in numerous cells. These features were principally maintained when grown as multicellular spheroids and in nude mice. More pronounced signs of differentiation (i.e., expression of cytokeratins 10 and 11, formation of glandular structures) were found in xenograft tumours. Independently, cytokeratins 13, 18 and 19 were detected in vitro and in vivo, reflecting the urothelial origin. The line forms distinct colonies in soft agar, expresses Lewis-x and Lewis-y antigens and reacts with monoclonal antibodies (MAbs) against CEA, β-HCG and URO-5. Cytogenetic analysis revealed several related clones with a rearrangement at chromosome I and loss of one X chromosome as common karyotypic changes in all clones. DNA content, as quantified by image analysis, showed a DNA stemline close to 2c. The new cell line HOK-I can be used as an in vitro model to study the mechanisms of heterogeneous differentiation patterns in bladder cancer.